2-Methylene-1Alpha-Hydroxy-19,21-Dinorvitamin D3 Analogs and Uses Thereof

ABSTRACT

Compounds of formula I are provided where X 1  and X 2  are independently selected from H or hydroxy protecting groups. Such compounds are used in preparing pharmaceutical compositions and are useful in treating a variety of biological conditions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional application60/744,383, filed Apr. 6, 2006, incorporated herein by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) and topharmaceutical formulations that include this compound. The inventionalso relates to the use of 2-methylene-1α-hydroxy-19,21-dinorvitamin D₃or salts thereof in the preparation of medicaments for use in treatingvarious diseases.

BACKGROUND OF THE INVENTION

The natural hormone, 1α,25-dihydroxyvitamin D₃ (also referred to as1α,25-dihydroxycholecalciferol and calcitriol) and its analog in theergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins, and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity is useful in thetreatment of a variety of diseases as established in the art, such asrenal osteodystrophy, vitamin D-resistant rickets, osteoporosis,psoriasis, and certain malignancies (see for example, Zemplar,Calcipotriol, MC-903, Dovonex, 22-oxa-1α,25-(OH)₂D₃) Slatopolsky, E.,Finch, J., Ritter, C., Denda, M., Morrissey, J., Brown, A. & DeLuca, H.(1995) Am. J. Kidney Dis. 26, 852-860; Kubodera, N., Sato, K. & Nishii,Y. (1997) in Vitamin D, eds. Feldman, D., Glorieux, F. H. & Pike, J. W.(Academic, New York), Vol. 63, pp. 1071-1086; Calverley, M. J. (1987)Tetrahedron Lett. 43, 4609-4619; Uskokovic, M. R., Studzinski, G. P. &Reddy, S. G. (1997) in Vitamin D, eds. Feldman, D., Glorieux, F. H. &Pike, J. W. (Academic, New York), Vol. 62, pp. 1045-1070; Kensler, T.W., Dolan, P. M., Gange, S. J., Lee, J.-K., Wang, Q. & Posner, G. H.(2000) Carcinogenesis 21, 1341-1345; Binderup, L., Binderup, E. &Godfredsen, W. O. (1997) in Vitamin D, eds. Feldman, D., Glorieux, F. H.& Pike, J. W. (Academic, New York), Vol. 61, pp. 1027-1043; Jones, G.(1997) in Vitamin D, eds. Feldman, D., Glorieux, F. H. & Pike, J. W.(Academic, New York), Vol. 58, pp. 973-994; Brown, A. J. & Slatopolsky,E. (1997) in Vitamin D, eds. Feldman, D., Glorieux, F. H. & Pike, J. W.(Academic, New York), Vol. 59, pp. 995-1009; Shankar, V. N., Propp, A.E., Schroeder, N. S., Surber, B. W., Makin, H. L. J. & Jones, G. (2001)Arch. Biochem. Biophys. 387, 297-306. All these references areincorporated herein by reference for all purposes.

As discussed above, renal osteodystrophy is a bone disease that occurswhen the kidneys fail to maintain the proper levels of calcium andphosphorus in the blood. Renal osteodystrophy is a common problem inpeople with kidney disease and affects 90 percent of dialysis patients.

Renal osteodystrophy is most serious in children because their bones arestill growing. The condition slows bone growth and causes deformities.One such deformity occurs when the legs bend inward toward each other oroutward away from each other; this deformity is referred to as “renalrickets.” Another important consequence is short stature. Symptoms canbe seen in growing children with renal disease even before they startdialysis.

The bone changes from renal osteodystrophy can begin many years beforesymptoms appear in adults with kidney disease. The symptoms of renalosteodystrophy are not usually seen in adults until they have been ondialysis for several years. Older patients and women who have gonethrough menopause are at greater risk for this disease because they'realready vulnerable to osteoporosis, even without kidney disease. If leftuntreated, the bones gradually become thin and weak, and a person withrenal osteodystrophy begins to experience bone and joint pain and anincreased risk of bone fractures.

In healthy adults, bone tissue is continually being remodeled andrebuilt. The kidneys play an important role in maintaining healthy bonemass and structure because it balances calcium and phosphorus levels inthe blood. If calcium levels in the blood become too low, theparathyroid glands release parathyroid hormone (PTH). This hormone drawscalcium from the bones to raise blood calcium levels. Too much PTH inthe blood causes disturbances in calcium and phosphorus homeostasis.This in turn removes too much calcium from the bones; over time, theconstant removal of calcium weakens the bones.

In this context, secondary hyperparathyroidism is characterized by anelevation of PTH associated with inadequate levels of active vitamin Dhormone. Typically, Vitamin D requires two sequential hydroxylations inthe liver and the kidney to bind to activate the Vitamin D receptor(VDR). The endogenous VDR activator, calcitriol [1,25(OH)₂ D₃] is ahormone that binds to VDRs that are present in the parathyroid gland,intestine, kidney, and bone to maintain parathyroid function and calciumand phosphorus homeostasis, and to VDRs found in many other tissues,including prostate, endothelium and immune cells. Phosphorus also helpsregulate calcium levels in the bones. Healthy kidneys remove excessphosphorus from the blood. When the kidneys stop working normally,phosphorus levels in the blood can become too high, leading to lowerlevels of calcium in the blood and resulting in the loss of calcium fromthe bones.

Healthy kidneys produce calcitriol to help the body absorb dietarycalcium into the blood and the bones. If calcitriol levels drop too low,PTH levels increase, and calcium is removed from the bones. Calcitrioland PTH work together to keep calcium balance normal and bones healthy.In a patient with kidney failure, the kidneys stop making calcitriol,dietary calcium is not absorbed and calcium is removed from the bones.

Controlling PTH levels prevents calcium from being withdrawn from thebones. Usually, overactive parathyroid glands are controllable with achange in diet, dialysis treatment, or medication. The drug cinacalcethydrochloride (Sensipar), approved by the Food and Drug Administrationin 2004, lowers PTH levels by binding to the calcium receptor thatcontrols PTH release. If PTH levels cannot be controlled, theparathyroid glands may need to be removed surgically. Other treatmentsfor the condition include taking synthetic calcitriol as a pill or in aninjectable form.

Renal osteodystrophy can also be treated with changes in diet. Reducingdietary intake of phosphorus is one of the most important steps inpreventing bone disease. Often, medications such as calcium carbonate(Tums), calcium acetate (PhosLo), sevelamer hydrochloride (Renagel), orlanthanum carbonate (Fosrenol) are prescribed with meals and snacks tobind phosphorus in the bowel, which decreases the absorption ofphosphorus into the blood.

Other treatment choices for renal osteodystrophy include Paricalcitol,the active ingredient of Zemplar (paracalcitol injection, USP), which isa synthetic, biologically active vitamin D analog of calcitriol withmodifications to the side chain and the A (19-nor) ring. Preclinical andin vitro studies have demonstrated that paricalcitol's actions aremediated through binding to the VDR, resulting in the selectiveactivation of Vitamin D response pathways. Calcitriol and paricalcitolhave been shown to reduce parathyroid hormone levels by inhibiting PTHsynthesis and secretion.

The structure of 1α,25-dihydroxyvitamin D₃ and the numbering system usedto denote the carbon atoms in this compound are shown below.

1α,25-Dihydroxyvitamin D₃=1α,25-Dihydroxycholecalciferol=Calcitriol

Typically, the class of vitamin D analogs such as 19-nor-vitamin Dcompounds is characterized by the absence of carbon 19 from the A-ringexocyclic methylene group, typical of the vitamin D system. Biologicaltesting of such 19-nor-analogs (e.g., 1α,25-dihydroxy-19-nor-vitamin D₃)revealed a selective activity profile with high potency in inducingcellular differentiation, and very low calcium mobilizing activity.Thus, these compounds are potentially useful as therapeutic agents forthe treatment of malignancies, or the treatment of various skindisorders. Two different methods of synthesis of such 19-nor-vitamin Danalogs have been described (Perlman et al., Tetrahedron Lett. 31, 1823(1990); Perlman et al., Tetrahedron Lett. 32, 7663 (1991), and DeLuca etal., U.S. Pat. No. 5,086,191).

In U.S. application Ser. Nos. 11/669,029 and 11/669,053 filed on Jan.30, 2007, (20R,25S)-2-methylene-19,26-dinor-1α,25-dihydroxyvitamin D3(NEL) and (20S,25S)-2-methylene-19,26-dinor-1α,25-dihydroxyvitamin D3(RAK) have been described and examined by DeLuca et al. as potentialdrugs for treatment of renal osteodystrophy. In U.S. Pat. No. 4,666,634,2β-hydroxy and alkoxy (e.g., ED-71) analogs of 1α,25-dihydroxyvitamin D₃have been described and examined by the Chugai group as potential drugsfor osteoporosis and as antitumor agents. See also Okano et al.,Biochem. Biophys. Res. Commun. 163, 1444 (1989). Other 2-substituted(with hydroxyalkyl, e.g., ED-120, and fluoroalkyl groups) A-ring analogsof 1α,25-dihydroxyvitamin D₃ have also been prepared and tested(Miyamoto et al., Chem. Pharm. Bull. 41, 1111 (1993); Nishii et al.,Osteoporosis Int. Suppl. 1, 190 (1993); Posner et al., J. Org. Chem. 59,7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

Various 2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ havealso been synthesized, i.e. compounds substituted at the 2-position withhydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with2-alkyl groups (DeLuca et al., U.S. Pat. No. 5,945,410), and with2-alkylidene groups (DeLuca et al., U.S. Pat. No. 5,843,928), whichexhibit interesting and selective activity profiles. All these studiesindicate that binding sites in vitamin D receptors can accommodatedifferent substituents at C-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs that are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while1α-hydroxy-2-methylene-19-nor-(20S)-homopregnacalciferol is described inU.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents. Other 19-norcompounds are disclosed in U.S. patent application Ser. Nos. 10/996,642and 10/997,698. All these patents and patent applications areincorporated herein by reference for all purposes.

Since the currently available treatments, including compounds andformulations described above have various limitations to a greater orlesser extent, new compounds and pharmaceutical formulations aredesirable that cure various diseases.

SUMMARY OF THE INVENTION

The invention generally provides2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) and relatedcompounds, pharmaceutical formulations that include SY-44 and the use ofthis compound in the preparation of medicaments for use in treatingvarious disease states.

Therefore, in one aspect, the invention provides a compound having theformula I as shown below:

where X₁ and X₂ are the same or different and are independently selectedfrom H or hydroxy-protecting groups. In some embodiments, X₁ and X₂ arehydroxy protecting groups such as silyl ether groups, alkyl ethergroups, alkoxyalkyl ether group, acetal groups and ester groups. In somesuch embodiments, X₁ and X₂ are t-butyldimethylsilyl ether group(TBDMS), trimethylsilyl ether group (TMS), triethylsilyl ether group(TES), Triisopropylsilyl ether group (TIPS), t-butyldiphenylsilyl ethergroup (TBDPS), tetrahydropyran group (THP), methoxyethoxymethyl group(MEM), methoxymethyl group (MOM), benzyl ether group, t-butyl ethergroup, N-phthalimido acetal group (Nphth), isopropylidene, trimethoxybutane, 2,4-dimethylpentan-3-yloxycarbonyl group (Doc). Various otherhydroxy protecting groups are known to one of ordinary skill in the art,for example see Jarowicki et al, J. Chem. Soc., Perkin Trans. 1, 1998,4005-4037, which is incorporated herein by reference for all purposes.

In other embodiments, X₁ and X₂ are H such that the compound is2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) having the formulaII as shown below:

Another embodiment of the present invention provides a pharmaceuticalcomposition, comprising an effective amount of the compound of formula Ior II and a pharmaceutically acceptable carrier. In this pharmaceuticalcomposition the effective amount comprises from about 0.01 μg to about 1mg of the compound per gram of the composition. More preferably, theeffective amount comprises from about 0.1 μg to about 500 μg of thecompound per gram of the composition.

In certain embodiments, the present invention provides a method oftreating a subject suffering from a biological condition, comprisingadministering an effective amount of the compound of formula I or II tothe subject, wherein the biological condition is selected from metabolicbone diseases, such as osteomalacia and vitamin D resistant rickets;psoriasis; leukemia; colon cancer; breast cancer; prostate cancer; skincancer; lung cancer; multiple sclerosis; lupus; diabetes mellitus; hostversus graft reaction; rejection of organ transplants; an inflammatorydisease selected from rheumatoid arthritis, asthma, or inflammatorybowel diseases selected from celiac disease, ulcerative colitis andCrohn's disease; a skin condition selected from wrinkles, lack ofadequate skin firmness, lack of adequate dermal hydration, orinsufficient sebum secretion; renal osteodystrophy; osteopenia; orosteoporosis particularly senile osteoporosis, postmenopausalosteoporosis, steroid-induced osteoporosis and low bone turnoverosteoporosis. In an exemplary embodiment, the biological condition isrenal osteodystrophy, vitamin D-resistant rickets, osteoporosis orpsoriatic arthritis. In another exemplary embodiment, the biologicalcondition is metabolic bone disease. In another exemplary embodiment,the biological condition is selected from leukemia, colon cancer, breastcancer, skin cancer, lung cancer, or prostate cancer. In yet anotherexemplary embodiment, the biological condition is selected from multiplesclerosis, lupus, diabetes mellitus, host versus graft reaction, orrejection of organ transplants. In still other exemplary embodiment, thebiological condition is selected from rheumatoid arthritis, asthma, orinflammatory bowel diseases selected from celiac disease, ulcerativecolitis and Crohn's disease. In yet other exemplary embodiment, thebiological condition is selected from wrinkles, lack of adequate skinfirmness, lack of adequate dermal hydration, or insufficient sebumsecretion.

Also preferably, in this embodiment, the effective amount of thecompound is administered orally, parenterally, transdermally, nasally,rectally, sublingually or topically to the subject. Yet more preferably,the effective amount of the compound is administered intraperitoneally.In this embodiment, the compound is administered in a dosage of from0.01 μg per day to 1 mg per day.

Another aspect of the invention provides the use of the compound offormula I in the preparation of a medicament for the treatment of abiological condition selected from metabolic bone diseases such asosteomalacia and vitamin D resistant rickets; psoriasis; leukemia; coloncancer; breast cancer; prostate cancer; skin cancer; lung cancer;multiple sclerosis; lupus; diabetes mellitus; host versus graftreaction; rejection of organ transplants; an inflammatory diseaseselected from rheumatoid arthritis, asthma, or inflammatory boweldiseases selected from celiac disease, ulcerative colitis and Crohn'sdisease; a skin condition selected from wrinkles, lack of adequate skinfirmness, lack of adequate dermal hydration, or insufficient sebumsecretion; renal osteodystrophy; osteopenia, or osteoporosis,particularly senile osteoporosis, postmenopausal osteoporosis,steroid-induced osteoporosis and low bone turnover osteoporosis.

Yet another preferred embodiment of the present invention provides thecompound having the formula II

The invention also teaches a pharmaceutical composition having aneffective amount of the compound of formula II and a pharmaceuticallyacceptable carrier.

Another aspect of the invention provides the use of the compound offormula II in the preparation of a medicament for the treatment of abiological condition selected from metabolic bone diseases such asosteomalacia and vitamin D resistant rickets; psoriasis; leukemia; coloncancer; breast cancer; prostate cancer; skin cancer; lung cancer;multiple sclerosis; lupus; diabetes mellitus; host versus graftreaction; rejection of organ transplants; an inflammatory diseaseselected from rheumatoid arthritis, asthma, or inflammatory boweldiseases selected from celiac disease, ulcerative colitis and Crohn'sdisease; a skin condition selected from wrinkles, lack of adequate skinfirmness, lack of adequate dermal hydration, or insufficient sebumsecretion; renal osteodystrophy; osteopenia; or osteoporosis,particularly senile osteoporosis, postmenopausal osteoporosis,steroid-induced osteoporosis and low bone turnover osteoporosis.

Further objects, features and advantages of the invention will beapparent from the following detailed description, drawings and appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 illustrate various biological activities of2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (referred to as “SY-44” inthe Figures) compared with those of the native hormone1α,25-dihydroxyvitamin D₃ (referred to as “1,25(OH)₂D₃” in the Figures).

FIG. 1 is a graph comparing the relative activity of SY-44 and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor.

FIG. 2 is a bar graph comparing the bone calcium mobilization activityof SY-44 with that of 1,25(OH)₂D₃.

FIG. 3 is a bar graph comparing the intestinal calcium transportactivity of SY-44 with that of 1,25(OH)₂D₃.

FIG. 4 is a graph comparing the percent HL-60 cell differentiation as afunction of the concentration of SY-44 with that of 1,25(OH)₂D₃.

FIG. 5 is a graph comparing the in vitro transcription activity of SY-44with that of 1,25(OH)₂D₃.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the invention provides a compound having the formula I asshown below:

where X₁ and X₂ are the same or different and are independently selectedfrom H or hydroxy-protecting groups. In some embodiments, X₁ and X₂ arehydroxy protecting groups such as silyl ether groups, alkyl ethergroups, alkoxyalyl ether group, acetal groups and ester groups. In somesuch embodiments, X₁ and X₂ are t-butyldimethylsilyl ether group(TBDMS), trimethylsilyl ether group (TMS), triethylsilyl ether group(TES), Triisopropylsilyl ether group (TIPS), t-butyldiphenylsilyl ethergroup (TBDPS), tetrahydropyran group (THP), methoxyethoxymethyl group(MEM), methoxymethyl group (MOM), benzyl ether group, t-butyl ethergroup, N-phthalimido acetal group (Nphth), isopropylidene,trimethoxybutane, 2,4-dimethylpentan-3-yloxycarbonyl group (Doc).Various other hydroxy protecting groups are known to one of ordinaryskill in the art, for example see Jarowicki et al, J. Chem. Soc., PerkinTrans. 1, 1998, 4005-4037, which is incorporated herein by reference forall purposes.

Also as used herein, the term “hydroxy-protecting group” signifies anygroup commonly used for the temporary protection of the hydroxy (—OH)functional group, such as, but not limited to, alkoxycarbonyl, acyl,alkylsilyl or alkylarylsilyl groups (hereinafter referred to simply as“silyl” groups), and alkoxyalkyl groups. Alkoxycarbonyl protectinggroups are alkyl-O—CO— groups such as methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term“acyl” signifies an alkanoyl group of 1 to 6 carbons, in all of itsisomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as anoxalyl, malonyl, succinyl, glutaryl group, or an aromatic acyl groupsuch as benzoyl, or a halo, nitro or alkyl substituted benzoyl group.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group. An extensive listof protecting groups for the hydroxy functionality is found inProtective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G. M.,John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) which can beadded or removed using the procedures set forth therein and which ishereby incorporated by reference in its entirety and for all purposes asif fully set forth herein.

A “protected hydroxy” group is a hydroxy group derivatized or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functional groups, e.g., the silyl, alkoxyalkyl,acyl or alkoxycarbonyl groups, as previously defined.

In other embodiments, X₁ and X₂ are H such that the compound is2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) having the formulaII as shown below:

The compound of formula II (SY-44) exhibits a desired, and highlyadvantageous, pattern of biological activity. This compound ischaracterized by high binding to vitamin D receptors, capability tostimulate transcription of reporter gene stably transfected in Ros17/2.8(bone) cells and capability to induce differentiation of HL-60 cells.Further, it has high intestinal calcium transport activity, as comparedto that of 1α,25-dihydroxyvitamin D₃, and has low ability to mobilizecalcium from bone, as compared to 1,25-dihydroxyvitamin D₃. Thus, it isuseful as a therapy for metabolic bone diseases. It may also be usefulas a therapy for osteopenia as well as suppression of secondaryhyperparathyroidism or renal osteodystrophy.

The compound of the invention is also especially suited for treatmentand prophylaxis of human disorders which are characterized by animbalance in the immune system, e.g. in autoimmune diseases, includingmultiple sclerosis, lupus, diabetes mellitus, host versus graftreaction, and rejection of organ transplants; and additionally for thetreatment of inflammatory diseases, such as rheumatoid arthritis,asthma, and inflammatory bowel diseases such as celiac disease,ulcerative colitis and Crohn's disease. Acne, alopecia and hypertensionare other conditions which are treated with the compound of theinvention.

The above compound is also characterized by relatively high celldifferentiation activity. Thus, this compound also provides atherapeutic agent for the treatment of psoriasis, or as an anti-canceragent, especially against leukemia, colon cancer, breast cancer, skincancer, lung cancer, and prostate cancer. In addition, due to itsrelatively high cell differentiation activity, this compound provides atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of this compound thus not only results in moisturizing ofskin but also improves the barrier function of skin.

The compounds of the invention are used to prepare pharmaceuticalformulations or medicaments that include a compound of the invention incombination with a pharmaceutically acceptable carrier. Suchpharmaceutical formulations and medicaments are used to treat variousbiological disorders such as those described herein. Methods fortreating such disorders typically include administering an effectiveamount of the compound or an appropriate amount of a pharmaceuticalformulation or a medicament that includes the compound to a subjectsuffering from the biological disorder. In some embodiments, the subjectis a mammal. In some such embodiments, the mammal is selected from arodent, a primate, a bovine, an equine, a canine, a feline, an ursine, aporcine, a rabbit, or a guinea pig. In some such embodiments, the mammalis a rat or is a mouse. In some embodiments, the subject is a primatesuch as, in some embodiments, a human.

The compounds is present in a composition to treat the above-noteddiseases and disorders in an amount from about 0.01 μg/gm to about 1mg/gm of the composition, preferably from about 0.1 μg/gm to about 500μg/gm of the composition, and is administered topically, transdermally,orally, or parenterally in dosages of from about 0.01 μg/day to about 1mg/day, preferably from about 0.1 g/day to about 500 μg/day.

In one embodiment, the invention provides compounds II as shown below:

In a preferred embodiment, 2-methylene-1α-hydroxy-19,21-dinorvitamin D₃(SY-44) was synthesized, and tested, and is useful in treating a varietyof biological conditions as described herein.

Preparation of 2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) canbe accomplished by condensing an appropriate bicyclic Windaus-Grundmanntype ketone (III) with the allylic phosphine oxide IV followed bydeprotection (removal of the Y₁ and Y₂ groups). Other compounds of thepresent invention are similarly synthesized.

In phosphine oxide IV, Y₁ and Y₂ are preferably hydroxy-protectinggroups such as silyl protecting groups. In a preferred embodiment, thetriethylsilyl group (TES) and t-butyldimethylsilyl (TBDMS) group areexamples of a particularly useful hydroxy-protecting groups. The processdescribed above represents an application of the convergent synthesisconcept, which has been applied effectively to the preparation ofnumerous vitamin D compounds (see Lythgoe et al., J. Chem. Soc. PerkinTrans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Toh etal., J. Org. Chem. 48, 1414 (1983); Baggiolini et al., J. Org. Chem. 51,3098 (1986); Sardina et al., J. Org. Chem. 51, 1264 (1986); J. Org.Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713; and DeLuca et al., U.S. Pat. No. 5,843,928all of which are hereby incorporated by reference in their entirety andfor all purposes as if fully set forth herein).

Phosphine oxide IV is a convenient reagent that can be used to prepare alarge number of 19-nor vitamin D compounds and is prepared according tothe procedures described by Sicinski et al., J. Med. Chem., 41, 4662(1998), DeLuca et al., U.S. Pat. No. 5,843,928; Perlman et al.,Tetrahedron Lett. 32, 7663 (1991); and DeLuca et al., U.S. Pat. No.5,086,191. Scheme I shows the general procedure for synthesizingphosphine oxide IV as outlined in U.S. Pat. No. 5,843,928 which ishereby incorporated by reference in its entirety as if fully set forthherein. Modification of the method shown in Scheme I is used to producea large number of vitamin D analogs as will be apparent to those skilledin the art. For example, a wide variety of phosphonium compounds is usedin place of the MePh₃P⁺ Br⁻ used to convert ketone B to alkene C.Examples of such compounds include EtPh₃P⁺ Br⁻, PrPh₃P⁺ Br⁻, andcompounds generally prepared by reaction of triphenylphosphine with analkyl halide, an alkenyl halide, a protected-hydroxyalkyl halide, and aprotected hydroxyalkenyl halide. Alkenes prepared using this proceduremay then be carried through to prepare a phosphine oxide in an analogousmanner to that used to prepare phosphine oxide H in Scheme I.Alternatively, an alkene analogous to compound C of Scheme I is reducedwith (Ph₃P)₃RhCl and H₂ to provide other vitamin D analogs. See U.S.Pat. No. 5,945,410 and Sicinski, R. R. et al., J. Med. Chem., 41,4662-4674 (1998) both of which are hereby incorporated by reference intheir entireties and for all purposes. Therefore, the procedure forforming the phosphine oxide shown in Scheme I is used to prepare a widevariety of vitamin D analogs in addition to the compound of the presentinvention.

Hydraindanones of structure III can prepared by known methods or adaptedmethods as will be readily apparent to one of skill in the art anddescribed herein. Specific examples of some important bicyclic ketonesused to synthesize vitamin D analogs are those described in Mincione etal., Synth. Commun 19, 723, (1989); and Peterson et al., J. Org. Chem.51, 1948, (1986).

In one preferred embodiment, ketone III (14) and compound of Formula II(SY-44) (17) were prepared by the following Scheme II, as shown below:

An overall process for synthesizing 2-alkylidene-19-nor-vitamin Dcompounds is illustrated and described in U.S. Pat. No. 5,843,928, U.S.Pat. No. 6,627,622, U.S. Pat. No. 6,579,861, U.S. Pat. No. 5,086,191,U.S. Pat. No. 5,585,369, and U.S. Pat. No. 6,537,981, which are herebyincorporated by reference in its entirety and for all purposes as iffully set forth herein.

Compounds of formula I and formula II can be prepared using the methodsshown in Schemes I, and II. For the compound of formula II, the startingmaterial, compound 10, was prepared using known procedures, as shownbelow in Scheme III. See also, Andrzej R. Daniewski and Wen Liu, J. Org.Chem. 66, 626-628 (2001), which is hereby incorporated by reference inits entirety and for all purposes as if fully set forth herein.

(i) TsCI, Et₃N, DMAP, CH₂Cl₂, 96%. (ii) LiBr, DMF, 55%. (iii) Ph₃P,PhMe, 81%.

Following examples illustrate synthesis and biological activity of thecompounds provided in the present invention. These Examples are forillustration purposes only and should not be deemed to limit the scopeof the invention.

EXAMPLE I SY-44 Synthesis Des-A,B-23,24-dinorcholane-8β,22-diol (1)

A solution of vitamin D₂ (5 g; 12.7 mmol) in methanol (400 mL) andpyridine (5 mL) was cooled to −78° C. while purging with argon. Theargon stream was stopped and stream of ozone was passed until blue colorappeared. The solution was purged with oxygen until blue colordisappeared and treated with NaBH₄ (1.2 g; 32 mmol). After 20 min. thesecond portion of NaBH₄ (1.2 g; 32 mmol) was added and reaction wasallowed to warm to room temperature. The third portion of NaBH₄ (1.2 g;32 mmol) was added and reaction mixture was stirred at room temperatureovernight. The reaction was quenched with 70 mL of water andconcentrated under vacuum. The residue was extracted with methylenechloride (3×100 mL). The organic phase was washed with 1M aqueoussolution of HCl (2×100 mL), saturated aqueous solution of NaHCO₃ (100mL), dried over anhydrous MgSO₄ and concentrated under vacuum. Theresidue was purified by flash chromatography (25% ethyl acetate/hexane)to yield 2.05 g (9.69 mmol; 76% yield) of diol 1 as white crystals.[α]_(D)=+56.0 (c 0.95, CHCl₃); m.p. 110÷111° C.; ¹H NMR (400 MHz, CDCl₃)δ 0.96 (3H, s), 1.03 (3H, d, J=6.6 Hz), 3.38 (1H, dd, J=10.5 Hz, J=6.8Hz), 3.64 (1H, dd, J=10.5 Hz, J=3.2 Hz), 4.09 (1H, d, J=2.3 Hz); ¹³C NMR(100 MHz, CDCl₃) δ 13.6, 16.6, 17.4, 22.6, 26.6, 33.5, 38.2, 40.2, 41.3,52.3, 52.9, 67.8, 69.2; MS (EI) m/z 212 (M⁺, 2), 194 (17), 179 (18), 163(10), 135 (19), 125 (34), 111 (100); exact mass calculated for C₁₃H₂₂O([M−H₂O]⁺) 194.1671, found 194.1665.

Des-A,B-22-(acetoxy)-23,24-dinorcholane-8β-ol (2)

To a stirred solution of 1 (3.50 g, 16.5 mmol) and DMAP (100 mg) intriethylamine (3.00 mL, 1.67 g, 21.6 mmol) and methylene chloride (300mL) acetic anhydride (1.54 mL, 2.18 g, 16.5 mmol) was added dropwise at0° C. The reaction mixture was kept at 4° C. overnight. Solvents wereremoved under reduced pressure and the residue was redissolved inmethylene chloride (200 mL), washed with 10% aqueous solution of HCl (50mL), saturated aqueous solution of NaHCO₃ (50 mL) and water (50 mL).Organic phase was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to give 4.06 g (16.0 mmol; 97% yield) of 2 as whitecrystals. [α]_(D)=+33.7 (c 0.90), CHCl₃); m.p. 78÷80° C.; ¹H NMR (500MHz, CDCl₃) □δ 0.96 (3H, s), 1.00 (3H, d, J=6.6 Hz), 2.05 (3H, s), 3.77(1H, dd, J=10.6 Hz, J=7.7 Hz), 4.06 (1H, dd, J=10.6 Hz, J=3.3 Hz), 4.11(1H, br s); ¹³C NMR (100 MHz, CDCl₃) δ 13.5, 17.0, 17.4, 21.0, 22.5,26.6, 33.5, 35.3, 40.2, 41.9, 52.3, 53.2, 69.1, 69.4, 171.4; MS (EI) m/z254 (M⁺, 2), 236 (5), 205 (2), 194 (12), 176 (22), 161 (14), 135 (16),125 (34), 111 (100); exact mass (ESI) calculated for C₁₅H₂₃O₃Na([M+Na]⁺) 277.1780, found 277.1791.

Des-A,B-22-(acetoxy)-8β-[(triethylsilyl)oxy]-23,24-dinorcholane (3)

To a stirred solution of 2 (4.00 g, 16.6 mmol) in methylene chloride (40mL) and 2,6-lutidine (2.67 mL, 2.46 g, 23.0 mmol) triethylsilyltrifluoromethanesulfonate (4.52 mL, 5.28 g, 20.0 mmol) was addeddropwise under argon at −50° C. After 30 min, wet methylene chloride (5mL) and water (80 mL) were added. The reaction mixture was extractedwith methylene chloride (3×120 mL) and organic phase was washed withsaturated aqueous solution of CuSO₄ (50 mL), dried over anhydrous Na₂SO₄and concentrated under reduced pressure to give crude 3 as oil.[α]_(D)=+42.2 (c 1.25, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.55 (6H, q,J=7.9 Hz), 0.93 (3H, s), 0.95 (9H, t, J=8.0 Hz), 0.98 (3H, d, J=6.6 Hz),2.05 (3H, s), 3.77 (1H, dd, J=10.6 Hz, J=7.5 Hz), 4.04-4.07 (2H, m); ¹³CNMR (125 MHz, CDCl₃) δ 4.9, 6.9, 13.5, 17.1, 17.6, 21.0, 23.0, 26.8,34.6, 35.4, 40.6, 42.2, 52.8, 53.4, 69.2, 69.6, 171.4; MS (EI) m/z 368(M⁺, 4), 339 (30), 325 (15), 177 (89), 145 (100); exact mass calculatedfor C₂₁H₄₀O₃Si 368.2747, found 368.2748.

Des-A,B-8-[(triethylsilyl)oxy]-23,24-dinorcholane-22-ol (4)

To a stirred solution of crude 3 in methanol (100 mL) 10% solution ofsodium methanolate in methanol (20 mL) was added dropwise. After 2 hsaturated aqueous solution of NH₄Cl (20 mL) and water (60 mL) were addedand the mixture was extracted with CH₂Cl₂ (5×100 mL). Organic phase wasdried over anhydrous Na₂SO₄, concentrated under reduced pressure and theresidue was purified on silica gel column (10÷20% ethyl acetate/hexane)to give 5.25 g (16.1 mmol; 97% yield from 2) of 4. [α]_(D)=+40.3 (c1.00, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.55 (6H, q, J=7.9 Hz),0.93-0.97 (12H, m), 1.02 (3H, d, J=6.6 Hz), 3.37 (1H, dd, J=10.4 Hz,J=6.8 Hz), 3.63 (1H, dd, J=10 Hz, J=3.0 Hz), 4.04 (1H, d, J=1.8 Hz); ¹³CNMR (100 MHz, CDCl₃) δ 4.9, 6.9, 13.6, 16.6, 17.6, 23.0, 26.8, 34.6,38.3, 40.6, 42.1, 52.8, 53.1, 68.0, 69.3; MS (EI) m/z 326 (M⁺, 10), 311(2), 297 (93), 283 (36), 225 (16), 193 (21), 177 (100); exact masscalculated for C₁₉H₃₈O₂Si 326.2641, found 326.2639.

Des-A,B-8β-[(triethylsilyl)oxy]-23,24-dinorcholane-22-al (5)

Sulfur trioxide pyridine complex (7.42 g, 46.5 mmol) was added to thestirred solution of 4 (2.32 g, 7.02 mmol) in triethylamine (5.46 mL,3.94 g, 39.0 mmol), anhydrous DMSO (8.0 mL) and anhydrous CH₂Cl₂ (40 mL)at 0° C. under argon. After 20 min. methylene chloride (150 mL) wasadded and reaction mixture was washed with saturated aqueous solution ofCuSO₄ (40 mL) and water (40 mL). Organic phase was dried over anhydrousNa₂SO₄, concentrated under reduced pressure and residue was purified onsilica gel (0.5÷2% ethyl acetate/hexane) to give 1.80 mg (5.56 mmol; 78%yield) of 5. [α]_(D)=+42.6 (c 1.15, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ0.57 (6H, q, J=7.9 Hz), 0.94-0.98 (12H, m), 1.10 (3H, d, J=6.8 Hz), 2.35(1H, m), 4.07 (1H, d, J=2.5 Hz), 9.58 (1H, d, J=3.2 Hz); ¹³C NMR (100MHz, CDCl₃) δ 5.0, 6.9, 13.4, 13.9, 17.6, 23.3, 26.2, 34.6, 40.6, 42.7,49.1, 51.8, 52.5, 53.2, 69.1, 205.3; MS (EI) m/z 324 (M⁺, 4), 311 (12),295 (100); exact mass calculated for C₁₇H₃₁O₂Si ([M−C₂H₅]⁺) 295.2093,found 295.2086.

Des-A,B-8β-[(triethylsilyl)oxy]-pregnane-20-one (6)

Through a solution of potassium tert-butanolate (3.7 g; 33 mmol) intert-butanol (90 mL) oxygen was passed for 15 min. Then a solution of 5in tert-butanol (45 mL) was added dropwise while purging with oxygen.Saturated aqueous solution of NH₄Cl (80 mL) and water (50 mL) were addedand the reaction products were extracted with Et₂O (5×150 mL). Organicphase was dried over anhydrous MgSO₄, concentrated under reducedpressure and the residue was purified by column chromatography (3÷6%ethyl acetate/hexane) to give 1.14 g (3.68 mmol; 67% yield) of 6.[α]_(D)=+107.1 (c 0.80, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.55 (6H, q,J=7.9 Hz), 0.85 (3H, s), 0.94 (9H, t, J=7.9 Hz), 2.09 (3H, s), 2.47 (1H,t, J=9.0 Hz), 4.07 (1H, d, J=2.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 4.9,6.9, 15.3, 17.6, 21.8, 23.1, 31.5, 34.4, 39.9, 43.7, 53.3, 64.5, 68.9,209.5; MS (EI) m/z 310 (M⁺, 50), 281 (84), 211 (73), 173 (94), 87 (100);exact mass calculated for C₁₈H₃₄O₂Si 310.2328, found 310.2332.

Des-A,B-8β-[(triethylsilyl)oxy]-testosterone acetate (7)

To a stirred solution of 6 in cyclohexane (50 mL) meta-chloroperbenzoicacid (77% max.; 1.5 g) was added at 0° C. Then the reaction mixture waswarmed up to room temperature and stirred for 5 days. Next portions ofmeta-chloroperbenzoic acid (1.0 g, 0.8 g and 0.6 g) were added after 1day, 2 days and 4 days, respectively. The suspension was filtered offand the filtrate was washed with saturated aqueous solution of NaHCO₃(20 mL). Organic phase was dried over anhydrous MgSO₄, concentratedunder reduced pressure and the residue was purified by columnchromatography (1÷3% ethyl acetate/hexane) to give 0.89 g (2.73 mmol;58% yield) of 7. [α]_(D)=+18.7 (c 0.9, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ0.56 (6H, q, J=7.9 Hz), 0.95 (9H, t, J=7.9 Hz), 1.11 (3H, s), 2.03 (3H,s), 4.05 (1H, d, J=2.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 4.9, 6.9, 13.6,17.2, 21.2, 22.2, 26.7, 34.5, 37.8, 42.0, 47.8, 69.0, 82.9, 171.3; MS(EI) m/z 326 (M⁺, 3), 297 (18), 283 (8), 145 (70), 135 (100); exact masscalculated for C₁₈H₃₄O₃Si 326.2277, found 326.2269.

Des-A,B-8-[(triethylsilyl)oxy]-testosterone (8)

7 (972 mg; 2.98 mmol) was dissolved in methanol (25 mL) and treated with10% solution of sodium metoxide in methanol (5 mL) for 2.5 h. Saturatedaqueous solution of NH₄Cl (10 mL) and water (15 mL) were added andproduct was extracted wit methylene dichloride (5×75 mL). Organic phasewas dried over anhydrous MgSO₄, concentrated under reduced pressure andthe residue was purified by column chromatography (5÷15% ethylacetate/hexane) to give 764 mg (2.69 mmol; 90% yield) of 8.[α]_(D)=+39.6 (c 0.95, CHCl₃); m.p. 95° C.; ¹H NMR (400 MHz, CDCl₃) δ0.56 (6H, q, J=7.9 Hz), 0.95 (9H, t, J=7.9 Hz), 0.96 (3H, s), 3.56 (1H,m), 4.02 (1H, d, J=2.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 5.0, 6.9, 12.4,17.3, 22.2, 29.9, 34.6, 37.5, 42.2, 48.1, 69.1, 82.2; MS (EI) m/z 284(M⁺, 9), 255 (100), 237 (40), 135 (42), 103 (66); exact mass calculatedfor C₁₄H₂₇O₂Si ([M−C₂H₅]⁺) 255.1780, found 255.1770.

Des-A,B-8β-[(triethylsilyl)oxy]-androstane-17-one (9)

To a stirred solution of 8 (760 mg; 2.68 mmol) and PPTS (30 mg; 0.12mmol) in methylene dichloride (90 mL) PDC (2.25 g; 5.98 mmol) was addedat 0° C. Cooling bath was removed and the reaction mixture was stirredfor 9 h. Then solvent was removed under reduced pressure and the residuewas purified by column chromatography (5÷10% ethyl acetate/hexane) togive 642 mg (2.28 mmol; 85% yield) of 9. [α]_(D)=+82.9 (c 0.90, CHCl₃);¹H NMR (500 MHz, CDCl₃) δ 0.60 (6H, q, J=7.9 Hz), 0.97 (9H, t, J=7.9Hz), 1.11 (3H, s), 2.43-2.47 (1H, m), 4.19 (1H, m); ¹³C NMR (125 MHz,CDCl₃) δ 4.9, 6.9, 16.3, 17.0, 21.3, 32.3, 34.5, 35.3, 47.5, 48.8, 69.9,221.2; MS (EI) m/z 282 (M⁺, 10), 252 (100), 133 (17), 103 (39); exactmass calculated for C₁₆H₃₀O₂Si 282.2015, found 282.2013.

(17Z)-Des-A,B-8β-[(triethylsilyl)oxy]-21-norcholest-17-ene (11)

To a stirred suspension of 10 (4.10 g; 9.35 mmol) in THF (13.5 mL) 1Msolution of potassium tert-butoxide in THF (8.90 mL; 8.90 mmol) wasadded dropwise at −10° C. The suspension was stirred and warmed up to 0°C. over 30 min. Then a solution of 9 (716 mg; 2.53 mmol) in THF (3.0 mL)was added via cannula and the resulting mixture was stirred at 45° C.for 4 days. Then saturated aqueous solution of NH₄Cl (20 mL) and water(30 mL) were added and the mixture was extracted with diethyl ether(3×100 mL). Organic phase was dried over anhydrous MgSO₄, concentratedunder reduced pressure and the residue was purified by columnchromatography (hexane÷5% ethyl acetate/hexane) to give 572 mg (1.60mmol; 63% yield) of 11 (Z/E ratio 5:1). [α]_(D)=+4.1 (c 0.95, CHCl₃); ¹HNMR (600 MHz, CDCl₃) δ 0.56 (6H, q, J=7.9 Hz), 0.86 (6H, d, J=6.7 Hz),0.95 (9H, t, J=7.9 Hz), 1.10 (3H, s), 4.11 (1H, s), 4.94 (0.83H, t,J=7.3 Hz), 5.36 (0.17H, t, J=4.7 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 5.0,7.0, 18.0, 19.9, 22.6, 22.7, 23.8, 27.5, 27.6, 27.7, 28.0, 28.7, 30.6,34.6, 38.3, 38.7, 38.9, 44.2, 52.8, 69.7, 119.3, 130.0, 149.9; MS (EI)m/z 364 (M⁺, 6), 335, (23), 321 (10), 279 (37), 232 (54), 205 (43), 171(51), 147 (100); exact mass calculated for C₂₃H₄₄OSi 364.3161, found364.3175.

Des-A,B-8β-[(triethylsilyl)oxy]-21-norcholestane (12)

A stirred mixture of 11 (552 mg; 1.52 mmol) and 5% Pd/C (160 mg) inethyl acetate (20 mL) was treated with hydrogen overnight. Then thecatalyst was filtered off and the filtrate was concentrated underreduced pressure. The residue was purified on silica gel Sep-Packcartridge (hexane) to give 515 mg (1.41 mmol; 93% yield) of 12.[α]_(D)=+41.8 (c 1.15, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.55 (6H, q,J=7.9 Hz), 0.81 (3H, s), 0.86 (6H, d, J=6.6 Hz), 0.95 (9H, t, J=7.9 Hz),4.05 (1H, m); ¹³C NMR (100 MHz, CDCl₃) δ 5.0, 7.0, 14.1, 17.6, 22.7,22.7, 23.4, 27.8, 28.0, 29.1, 30.0, 35.0, 38.9, 39.1, 41.5, 51.7, 52.8,69.2; MS (EI) m/z 337 (100), 323 (59), 271 (67), 233 (77); exact masscalculated for C₂₁H₄₁OSi ([M−C₂H₅]⁺) 337.2927, found 337.2911.

Des-A,B-21-norcholestane-8β-ol (13).

To a stirred solution of 12 (485 mg; 1.33 mmol) in n-butanol (25 mL)(1S)-(+)-10-camphorsulfonic acid (330 mg; 1.42 mmol). After 1 daysolvent was removed under reduced pressure and the residue was purifiedby column chromatography (5÷15% ethyl acetate/hexane) to give 328 mg(1.30 mmol; 98% yield) of 13. [α]_(D)=+34.1 (c 1.25, CHCl₃); ¹H NMR (400MHz, CDCl₃) δ 0.84 (3H, s), 0.86 (6H, d, J=6.6 Hz), 4.10 (1H, br s); ¹³CNMR (100 MHz, CDCl₃) δ 14.0, 17.4, 22.7, 22.7, 22.9, 27.6, 28.0, 28.0,29.0, 29.9, 33.9, 38.5, 39.1, 41.3, 51.5, 52.3, 69.3; MS (EI) m/z 252(M⁺, 43), 237 (44), 219 (22), 209 (19), 125 (49), 111 (100); exact masscalculated for C₁₇H₃₂O 252.2453, found 252.2446.

Des-A,B-21-norcholestane-8-one (14)

To a stirred solution of 13 (10 mg; 35 μmol) and PPTS (2 crystals) inmethylene dichloride (3 mL) PDC (55 mg; 145 μmol) was added at 0° C.Cooling bath was removed and the mixture was stirred for 3 h. Thensolvent was removed under reduced pressure and the residue was purifiedon silica gel Sep-Pack cartridge (2-5% ethyl acetate/hexane) to give 8mg (32 μmol; 80% yield) of 14. ¹H NMR (400 MHz, CDCl₃) δ 0.55 (3H, s),0.87 (6H, d, J=6.6 Hz), 2.42 (1H, dd, J=11.2 Hz, J=7.7 Hz); ¹³C NMR (100MHz, CDCl₃) δ 12.7, 19.6, 22.6, 22.7, 24.0, 27.8, 27.8, 28.0, 28.9,30.2, 37.2, 39.1, 41.1, 49.7, 51.6, 61.5, 211.9; MS (EI) m/z 250 (M⁺,94), 235 (94), 207 (92), 159 (84), 152 (100); exact mass calculated forC₁₇H₃₀O 250.2297, found 250.2292.

2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) (17)

To a stirred solution of 15 (20 mg; 35 μmol) in THF (300μ) 10 □L of 1.2M solution of PhLi in cyclohexane/ether (7/3) was added at −20° C. untilthe solution became deep orange. Then 27 μL (32 μmol) of the solution ofPhLi was added drop wise. After 20 min. the reaction mixture was cooleddown to −78° C. and the solution of 14 (7 mg; 28 μmol) in THF (150 μL)was added via cannula. After 3 h cooling bath was removed and thereaction mixture was stirred at 4° C. overnight. Then ethyl acetate (10mL) was added and organic phase was washed with brine (2 mL), dried overanhydrous MgSO₄ and concentrated under reduced pressure. The residue waspurified on silica gel Sep-Pack cartridge (hexane÷2% ethylacetate/hexane) to give 5 mg (8 μmol) of crude 16.

To a stirred solution of 16 (5 mg; 8 μmol) in n-butanol (700 μL)(1S)-(+)-10-camphorsulfonic acid (5 mg; 22 μmol) was added at 0° C. Thencooling bath was removed and the reaction mixture was stirred for 1week. A few drops of saturated aqueous solution of NaHCO₃ and water (2mL) were added and the mixture was extracted with ethyl acetate (3×5mL). Combined organic phases were dried over anhydrous MgSO₄,concentrated under reduced pressure and the residue was purified onsilica gel Sep-Pack cartridge (20-30% ethyl acetate/hexane) to give 1 mg(3 μmol; 11% yield from 14) of 17. UV (EtOH) λ_(max)=243, 251, 261 nm;¹H NMR (600 MHz, CDCl₃) δ 0.45 (3H, s), 0.87 (6H, d, J=6.6 Hz),2.28-2.36 (2H, m), 2.58 (1H, m), 2.82-2.87 (2H, m), 4.45-4.51 (2H, m),5.09 (1H, s), 5.11 (1H, s), 5.88 (1H, d, J=11.3 Hz), 6.36 (1H, d, J=11.3Hz).

Toluene-4-sulfonic acid 5-methyl-hexyl ester (18)

To a stirred solution of 5-methyl-1-hexanol (3.65 mL; 3.00 g; 25.7mmol), triethylamine (5.00 mL; 3.64 g; 36.0 mmol) and DMAP (200 mg; 1.64mmol) in methylene dichloride (120 mL) tosyl chloride (5.72 g; 30.0mmol) was added at 0° C. Then cooling bath was removed and the mixturewas allowed to stand overnight. Saturated aqueous solution of NH₄Cl (30mL) and water (30 mL) were added and the mixture was extracted withmethylene dichloride (3×150 mL). Combined organic phases were dried overanhydrous MgSO₄ and concentrated under reduced pressure. The residue waspurified by column chromatography (5-10% ethyl acetate/hexane) to give6.66 g (24.7 mmol; 96% yield) of 22. ¹H NMR (400 MHz, CDCl₃) δ 0.83 (6H,d, J=6.6 Hz), 1.09 (2H, m), 1.28 (2H, m), 1.47 (1H, m), 1.62 (2H, m),2.45 (3H, s), 4.02 (2H, t, J=6.5 Hz), 7.34 (2H, d, J=8.1 Hz), 7.79 (2H,d, J=8.1 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 21.6, 22.5, 23.2, 27.8, 29.1,38.2, 70.2, 127.9, 129.8, 133.2, 144.6; MS (EI) m/z 255 (2), 226 (2),205 (3), 190 (5), 173 (67), 155 (57), 98 (59), 91 (100); exact masscalculated for C₁₃H₁₉O₃S ([M−CH₃]⁺) 255.1055, found 255.1062.

1-Bromo-5-methyl-hexane (19)

To a stirred solution of LiBr (6.26 g; 72.0 mmol) in DMF (40 mL) asolution of 18 (6.60 g; 24.4 mmol) in DMF (6 mL) was added. Theresulting mixture was stirred at 45° C. for 3 h. Then water (100 mL) wasadded and the reaction product was extracted with diethyl ether (5×250mL). Removal of solvent yielded in 2.40 g (13.4 mmol; 55%) of 19. ¹H NMR(400 MHz, CDCl₃) δ 0.88 (6H, d, J=6.6 Hz), 1.19 (2H, m), 1.42 (2H, m),1.54 (1H, m), 1.83 (2H, m), 3.41 (2H, t, J=6.9 Hz); ¹³C NMR (100 MHz,CDCl₃) δ 22.5, 26.0, 27.8, 33.1, 34.1, 38.0; MS (EI) m/z 179 (M⁺, 80),163 (14), 155 (9), 137 (100); exact mass calculated for C₆H₁₂Br([M−CH₃]⁺) 163.0122, found 163.0121.

(5-methylhexyl)triphenylphosphonium bromide (10)

A solution of 19 (2.30 g; 12.8 mmol) and triphenylphosphine (3.70 g;14.1 mmol) was refluxed in toluene (12 mL) for 20 h. Then solvent wasremoved and the resulting crystal was washed with toluene (3 mL) anddiethyl ether (3 mL) to give 4.57 g (10.4 mmol; 81% yield) of 10. m.p.227-228° C.; ¹H NMR (500 MHz, CD₃CN) δ 0.82 (6H, d, J=6.6 Hz), 1.16 (2H,m), 1.42-1.52 (3H, m), 1.59 (2H, m), 3.30 (2H, m), 7.72 (12H, m), 7.85(3H, m); ¹³C NMR (125 MHz, CD₃CN) δ 22.7, 23.2, 28.4, 28.8, 28.9, 38.6,131.2, 131.3, 134.7, 134.7, 136.0.

EXAMPLE II Biological Activity

(A) Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wasoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(3H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of <10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

(B) HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (<0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

(C) In vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydyroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.(RLU=relative luciferase units)

(D) Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (Suda et al.,Purified Rodent Diet-Diet 11, containing 0.47% and 0.3% phosphorus andfat soluble vitamins A, D, E and K are provided ad libitum) (0.47% Ca)diet+AEK for one week followed by Diet 11 (0.02% Ca)+AEK for 3 weeks.The rats were then switched to a diet containing 0.47% Ca for one weekfollowed by two weeks on a diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined as a measure of bone calciummobilization. The first 10 cm of the intestine was also collected forintestinal calcium transport analysis using the everted gut sac method.The everted gut sac assay was carried out as previously described byMartin and Deluca, Am. J. Physiol. 216, 1351 (1969); (DeLuca et al.,U.S. Pat. No. 4,188,345), which are incorporated by reference as iffully set forth herein.

Dose Preparation

Control Material

A. Negative Control Material

The negative control material is prepared by volumetrically measuringethanol (<5%) and propylene glycol, mixing, and then placing in storageat 2 to 8° C.

B. Positive Control Material

1,25(OH)₂D₃ is prepared by determining the concentration of an ethanolstock solution using UV spectrophotometry (extinctioncoefficient=18,200; λ_(max)=265 nm). The required amount of 1,25(OH)₂D₃is volumetrically measured into propylene glycol so that there was lessthan 5% ethanol in the final solution. The solution is mixed and thenstored at 2 to 8° C.

Test Material

The analogs are prepared by first determining the concentration of anethanol stock solution using UV spectrophotometry (extinctioncoefficient=42,000; λ_(max)=252 nm). The analog solutions are thenvolumetrically added to propylene glycol so that there was less than 5%ethanol in the final solution. The solution is mixed and stored at 2 to8° C.

Serum Calcium Analysis

Twenty-four hours after the final dose, approximately 1 ml of blood iscollected from the tail artery of each experimental animal. The blood isallowed to coagulate at room temperature and then centrifuged at 3000×gfor 15 minutes. The serum is transferred to a polypropylene tube andstored frozen at −20° C. The level of calcium is determined by dilutingthe serum into 0.1% lanthum chloride and measuring the absorbance on anatomic absorption spectrophotometer (Perkin Elmer Model 3110, Shelton,Conn.).

2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) binds to therecombinant vitamin D receptor, and is 10 times less active whencompared to 1α,25-dihydroxyvitamin D₃ in this respect (see FIG. 1).Additionally, it is also 10 times less active in stimulatingtranscription of a reporter gene stably transfected in Ros17/2.8 (bone)cells (see FIG. 5). It is also slightly less active as1α,25-dihydroxyvitamin D₃ in inducing differentiation of HL-60 cells(see FIG. 4). It has less bone calcium mobilization activity than does1α,25-dihydroxyvitamin D₃ (see FIG. 2). It has higher intestinal calciumtransport activity than does 1α,25-dihydroxyvitamin D₃ (see FIG. 3).Accordingly, SY-44 is expected to possess significant activity insuppressing parathyroid hormone levels in normal rats.

Similarly, other similar compounds of the present invention as shown informula I or II, are expected to bind to the vitamin D receptor,stimulate transcription of a reporter gene stably transfected in Ros17/2.8 (bone) cells, induce differentiation of HL-60 cells, have limitedcalcemic activity when measured either by intestinal calcium transportor bone calcium mobilization than 1α,25-dihydroxyvitamin D₃ and possesssignificant activity in suppressing parathyroid hormone levels.

Accordingly, this compound SY-44 and other compounds described in theinvention should find its uses in the treatment of autoimmune diseasessuch as multiple sclerosis, type I diabetes, rheumatoid arthritis,lupus, and other similar degenerative diseases. It should also havesignificant activity in treating malignant growth such as colorectal,breast, skin, lung and prostate cancers. This compound should also beuseful in treating secondary hyperparathyroidism found in patients whohave lost kidney function such as those on hemodialysis or peritonealdialysis. It may also be useful in treating osteopenia as well asmetabolic bone diseases such as osteomalacia, vitamin D resistantrickets and osteoporosis particularly senile osteoporosis,postmenopausal osteoporosis, steroid-induced osteoporosis and low boneturnover osteoporosis because of its significant activity in intestinalabsorption of calcium.

In one embodiment, the compounds of formula I are used in apharmaceutical composition. For example, each ml of the pharmaceuticalcomposition may comprise 5 μg of the compound, 30% (v/v) propyleneglycol and 20% (v/v) alcohol.

The compounds of the invention are also useful in preventing or treatingobesity, inhibiting adipocyte differentiation, inhibiting SCD-1 genetranscription, and/or reducing body fat in animal subjects. Therefore,in some embodiments, a method of preventing or treating obesity,inhibiting adipocyte differentiation, inhibiting SCD-1 genetranscription, and or reducing body fat in animal subject includesadministering to the animal subject, an effective amount of the compoundor a pharmaceutical composition that includes the compound.Administration of the compound or the pharmaceutical composition to thesubject inhibits adipocyte differentiation, inhibits gene transcription,and/or reduces body fat in the animal subject.

For treatment purposes, the compounds defined by formula I and formulaII are formulated for pharmaceutical applications as a solution ininnocuous solvents, or as an emulsion, suspension or dispersion insuitable solvents or carriers, or as pills, tablets or capsules,together with solid carriers, according to conventional methods known inthe art. Any such formulations may also contain other pharmaceuticallyacceptable and non-toxic excipients such as stabilizers, anti-oxidants,binders, coloring agents or emulsifying or taste-modifying agents.Pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is hereby incorporated by reference in its entirety and for allpurposes as if fully set forth herein.

The compounds are administered orally, topically, parenterally, nasally,rectally, sublingually or transdermally. The compounds areadvantageously administered by injection or by intravenous infusion orsuitable sterile solutions, or in the form of liquid or solid doses viathe alimentary canal, or in the form of creams, ointments, patches, orsimilar vehicles suitable for transdermal applications. In someembodiments, doses of from 0.001 μg to about 1 mg per day of thecompound are appropriate for treatment purposes. In some suchembodiments an appropriate and effective dose may range from 0.01 μg to1 mg per day of the compound. In other such embodiments an appropriateand effective dose may range from 0.1 μg to 500 μg per day of thecompound. Such doses will be adjusted according to the type of diseaseor condition to be treated, the severity of the disease or condition,and the response of the subject as is well understood in the art. Thecompound is suitably administered alone, or together with another activevitamin D compound.

In one embodiment, the compound of formula II is used in apharmaceutical composition. For example, each ml of the pharmaceuticalcomposition may comprise 5 μg of the compound, 30% (v/v) propyleneglycol and 20% (v/v) alcohol.

Compositions for use in the invention include an effective amount of(2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ (SY-44) as the activeingredient, and a suitable carrier. An effective amount of the compoundfor use in accordance with some embodiments of the invention willgenerally be a dosage amount such as those described herein, and isadministered topically, transdermally, orally, nasally, rectally,sublingually or parenterally. In one embodiment, the dosage isadministered intraperitoneally.

The compounds of formula I or II are advantageously administered inamounts sufficient to effect the differentiation of promyelocytes tonormal macrophages. Dosages as described above are suitable, it beingunderstood that the amounts given are to be adjusted in accordance withthe severity of the disease, and the condition and response of thesubject as is well understood in the art.

The compound is formulated as creams, lotions, ointments, aerosols,suppositories, topical patches, pills, capsules or tablets, or in liquidform as solutions, emulsions, dispersions, or suspensions inpharmaceutically innocuous and acceptable solvent or oils, and suchpreparations may contain, in addition, other pharmaceutically innocuousor beneficial components, such as stabilizers, antioxidants,emulsifiers, coloring agents, binders or taste-modifying agents.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationis in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration is in the form of a suppositoryincorporating the active ingredient and carrier such as cocoa butter, orin the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100 microns.

The formulations may conveniently be presented in dosage unit form andis prepared by any of the methods well known in the art of pharmacy. Bythe term “dosage unit” is meant a unitary, i.e., a single dose which iscapable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

All references cited herein are specifically incorporated by referencein their entireties and for all purposes as if fully set forth herein.

It is understood that the invention is not limited to the embodimentsset forth herein for illustration, but embraces all such forms thereofas come within the scope of the following claims.

1. A compound having the formula I

wherein X₁ and X₂ are independently selected from H and hydroxyprotecting groups.
 2. The compound of claim 1, wherein X₁ and X₂ arehydroxy protecting groups.
 3. The compound of claim 2, wherein X₁ and X₂are triethylsilyl or t-butyldimethylsilyl groups.
 4. The compound ofclaim 1, wherein X₁ and X₂ is H and the compound has the formula II


5. A pharmaceutical composition, comprising an effective amount of thecompound of claim 1 and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5 wherein the effective amountcomprises from about 0.01 μg to about 1 mg of the compound per gram ofthe composition.
 7. The pharmaceutical composition of claim 5 whereinthe effective amount comprises from about 0.1 μg to about 500 μg of thecompound per gram of the composition.
 8. A method of treating a subjectsuffering from a biological condition, comprising administering aneffective amount of the compound of claim 1 to the subject, wherein thebiological condition is selected from a metabolic bone disease;psoriasis; leukemia; colon cancer; breast cancer; prostate cancer; skincancer; lung cancer; multiple sclerosis; lupus; diabetes mellitus; hostversus graft reaction; rejection of organ transplants; an inflammatorydisease selected from rheumatoid arthritis, asthma, or inflammatorybowel diseases; a skin condition selected from wrinkles, lack ofadequate skin firmness, lack of adequate dermal hydration, orinsufficient sebum secretion; renal osteodystrophy; osteopenia; orosteoporosis.
 9. The method of claim 8, wherein the biological conditionis renal osteodystrophy, vitamin D-resistant rickets, osteoporosis orpsoriatic arthritis.
 10. The method of claim 8, wherein the biologicalcondition is selected from leukemia, colon cancer, breast cancer, skincancer, lung cancer or prostate cancer.
 11. The method of claim 8,wherein the biological condition is selected from multiple sclerosis,lupus, diabetes mellitus, host versus graft reaction, or rejection oforgan transplants.
 12. The method of claim 8, wherein the biologicalcondition is selected from rheumatoid arthritis, asthma, or inflammatorybowel diseases selected from celiac disease, ulcerative colitis andCrohn's disease.
 13. The method of claim 8, wherein the biologicalcondition is selected from wrinkles, lack of adequate skin firmness,lack of adequate dermal hydration, or insufficient sebum secretion. 14.The method of claim 8 wherein the biological condition is metabolic bonedisease.
 15. The method of claim 8, wherein the compound is administeredorally, parenterally, nasally, rectally, sublingually, transdermally ortopically to the subject.
 16. The method of claim 8, wherein thecompound is administered intraperitoneally.
 17. The method of claim 8,wherein the compound is administered in a dosage of from 0.01 μg per dayto 1 mg per day.
 18. A compound having the formula II


19. A pharmaceutical composition, comprising an effective amount of thecompound of claim 18 and a pharmaceutically acceptable carrier.
 20. Thepharmaceutical composition of claim 19 wherein the effective amountcomprises from about 0.01 μg to about 1 mg of the compound per gram ofthe composition.
 21. The pharmaceutical composition of claim 19 whereinthe effective amount comprises from about 0.1 μg to about 500 μg of thecompound per gram of the composition.
 22. A method of treating orpreventing obesity of an animal, inhibiting adipocyte differentiation,inhibiting SCD-1 gene transcription, and/or reducing body fat in ananimal comprising administering to an animal in need thereof aneffective amount fo a compound having the formula:

wherein X₁ and X₂ are independently selected from H and hydroxyprotecting groups.
 23. The method of claim 22, wherein the compound isadministered orally, parenterally, nasally, rectally, sublingually,transdermally or topically to the animal.
 24. The method of claim 22,wherein the compound is administered in a dosage of from 0.01 μg per dayto 1 mg per day.
 25. The method of claim 22, wherein the compound is2-methylene-1α-hydroxy-19,21-dinorvitamin D₃ having the formula:


26. The method of claim 22, wherein the animal is a human.
 27. Themethod of claim 22, wherein the animal is a domestic animal.
 28. Themethod of claim 22, wherein the animal is an agricultural animal.